Previously, in order to produce an objective substance at a high production rate by a bio procedure, many attempts have been tried to enhance expression of a catalyst enzyme gene involved in any one of metabolic pathways of microorganisms to an objective substance. It is identified that a dicarboxylic acid intervening in the tricarboxylic acid cycle such as succinic acid and the like is produced by incorporating carbonate ion from phosphoenolpyruvic acid or pyruvic acid produced in a glycolytic pathway of saccharides through a catalytic action of phosphoenolpyruvate carboxylase (hereinafter, referred to as PEPC) or pyruvate carboxylase (hereinafter, referred to as PC), wherein a reductive tricarboxylic acid cycle reaction via oxaloacetic acid takes place.
Based on above mentioned metabolic pathways, as a process for producing organic acid such as malic acid, fumaric acid, succinic acid and the like using a coryneform bacterium, a method of recombining a PEPC gene (Japanese Patent Application Laid-Open No. 11-196887) and a method of recombining a PC gene (Japanese Patent Application Laid-Open No. 11-196888) have been proposed.
However, the production rate of organic acid such as succinic acid and the like by any of these methods is not sufficient, and further improvement is needed.
On the other hand, there has been proposed a technique of simultaneously producing succinic acid, acetic acid and ethanol using an Escherichia coli mutant deficient in a lactate dehydrogenase (hereinafter, referred to as ldh) gene (enzyme gene involved in production route of lactic acid from pyruvic acid) which is one of the essential features of the present invention (U.S. Pat. No. 5,770,435, U.S. Pat. No. 5,869,301). The Escherichia coli mutant used in these US patents is an Escherichia coli mutant (AFP 111 strain) obtained by transforming an Escherichia coli strain (NZN 111 strain) deficient in an ldh gene and a pfl gene (pyruvate formate-lyase gene) so as to be capable of producing simultaneously succinic acid, acetic acid and ethanol, by mutation treatment (a mutated expression gene is not expressly described) or recombination treatment (treatment by introduction of a plasmid containing malate dehydrogenase-mdh-gene) under the anaerobic condition.
These two US patents have the similar technical contents by the same inventors. Although the present invention and these two US patents use respectively different organism species, but, similarly deficient in ldh gene expression function which is an essential feature of the present invention, the contents of metabolism function of a microorganism to be transformed are entirely different in view of the technical contents and results based on respective inventive concepts.
The difference in metabolism function between a coryneform bacterium transformant used in the present invention and the aforementioned Escherichia coli mutant is clear from the following two facts.
1) The Escherichia coli mutant (AFP 111) is further transformed by mutation or recombination of the Escherichia coli strain (NZN 111 strain) which is impossible in anaerobic growth because of deficiency in both genes of an ldh gene and a pfl gene, so as to have ethanol production function.
But a recombinant coryneform bacterium used in the present invention has not ethanol production function at all.
2) The Escherichia coli mutant (AFP 111) has not an essential requirement for the supply of carbonate ion in the production of succinic acid from saccharides (related with the above 1, since AFP 111 has an ethanol- and acetic acid-producing function, it is presumed that carbon dioxide gas produced as a byproduct of these fermentation products is utilized). In the production of dicarboxylic acids such as succinic acid and the like from saccharides by a coryneform bacterium of the present invention, it is essential to supply carbonate or bicarbonate ions or carbon dioxide gas from the outside.
Like this, it is thought that the Escherichia coli mutant and a coryneform bacterium of the present invention which are similarly deficient in an ldh gene have the entirely different metabolism functions and mechanisms. It is clear that the technique of the present invention is not obtained by combining two US patents regarding an Escherichia coli mutant (AFP 111) (in the present invention, mutation treatment and recombination technique of these US patents are not used) and the technique of the two Japanese Laid-Open Publications regarding a coryneform bacterium, and technical constitutional contents thereof are different.
Regarding production of succinic acid, the ldh gene-disrupted effect is made clear in Japanese Patent Application National Publication (Laid-Open) No. 2002-511250. In this published patent application, succinic acid production behavior is investigated regarding each of an ldh gene-disrupted mutant of an Escherichia coli wild strain (MG1655) and a double recombinant of PC gene high expression (although Escherichia coli wild strain has not originally a PC gene, introduction of a foreign PC gene is also called “high expression”)/ldh gene disruption (see Japanese Patent Application National Publication (Laid-Open) No. 2002-511250, Example II, Table 4).
In the same Table, it is shown that disruption of an ldh gene of an Escherichia coli wild strain hardly influences on succinic acid production (has little effect), and disruption of an ldh gene exhibits an inhibitory effect on the potentiation of succinic acid production by high expression of a PC gene.
These results show that although disruption, inhibition or interruption of a pathway other than a pathway of metabolism from pyruvic acid to succinic acid (e.g. pathway of production of lactic acid from pyruvic acid) in order to potentiate the flow of a carbon substance in the metabolic pathway from pyruvic acid to an objective substance (succinic acid) could be expected to have an effect of accumulating pyruvic acid flowing to an objective substance (effect of increasing amount of substrate), and such transformations are thought as if they are an easily thinkable means, the fact is converse. That is, it is shown that disruption of an ldh gene cannot be said to primarily lead to positive effect to succinic acid production.